Numerous studies showed that lipid transfer proteins (LTPs) play important roles in flower, development, cuticular wax deposition and pathogen responses; however, their roles in abiotic stresses are relatively less reported. This study characterized the function of a maize LTP gene (ZmLTP3) during drought stress. ZmLTP3 gene was transferred into maize inbred line Jing2416; subsequently the glyphosate and drought tolerance of the over-expression (OE) lines were analyzed. Analysis showed that OE lines could significantly enhance drought tolerance. Transgenic maize lines OE6, OE7 and OE8 showed lower cell membrane damage, higher chlorophyll contents, higher protective enzymes activities, better growth and development under drought condition. The results strongly indicated that overexpression of ZmLTP3 could increase drought tolerances in maize.